Cells within a tissue are autonomous units, yet constantly depend on signals from their surrounding. This information exchange is mediated by membrane proteins that assemble into localized, spatial and temporal organized multi-unit complexes. We will investigate on three levels how cells regulate subunit protein biosynthesis, complex assembly, and distribution of subunits within the complex. We use gap junctions as a model system. Gap junction channels cluster together comparable to claudins, cadherins, integrins, and acetylcholine receptors that are central to tight junctions, adherens junctions, desmosomes, hemidesmosomes, focal adhesions, and electrical synapses. We want to determine, especially in cells that co-express more than one connexin isoform (the gap junction channel subunit proteins) where exactly connexins are synthesized, where they begin to interact, how they are trafficked to the plasma membrane, how cells build the channel cluster, and find helper proteins that aid in these processes (aim I). Furthermore, we want to identify specific signals presumably located within the connexin polypeptide sequences that control subunit compatibility and channel composition; and determine whether aberrant connexin interactions can lead to disease (aim II). Finally, we want to find out how cells control the distribution of channels within a cluster (aim III). The approach includes to construct and express fluorescence tagged connexins, amino acid exchange variants, disease mutants, and chimeras in novel cell-free, cell-based, and live-cell systems that we have developed, track connexins in living cells by high-resolution, multi-color time lapse microscopy, determine their interaction, and use biochemical and immunological approaches to characterize helper proteins and probe channel environments. Results will provide significant novel insight into the biosynthesis, subunit assembly, composition, and function of gap junctions and of other oligomeric membrane structures; they will elucidate mechanistic principles of connexin related diseases; and they will aid in understanding the dynamic structural composition of other localized membrane signaling.